Vaccine based on a cellular penetration factor from an apicomplexan parasite

ABSTRACT

An antigenic component, for use in a vaccine capable of promoting, production, in a subject of an antibody specific to the antigenic component, which antibody is capable of specifically binding to the Pid protein having the amino acid sequence in Seq ID No. 1.

This application is the National Phase of International ApplicationPCT/GB01/04985 filed Nov. 9, 2001 which designated the U.S. and thatInternational Application was published under PCT Article 21(2) inEnglish.

FIELD OF THE INVENTION

This invention relates to an antigenic component for use in a vaccine,particularly for diseases such as those caused by apicomplexanparasites.

BACKGROUND TO THE INVENTION

The apicomplexans comprise a range of parasites including those of thegenera: Eimeria; Isospora; Toxoplasma; Hammondia; Cystoisospora;Sarcocystis; Besnoitia; Frenkelia; Cryptosporidia; Plasmodia; Babesia;and Theileria.

All genera of the apicomplexans have a specialized organelle called theapical complex (hence their name). This organelle contains secretorygranules/proteins that are extruded onto the surface of target cellsduring invasion. Extrusion of these proteins precedes cell entry

These parasites are associated with disease in a wide variety of hostorganisms. Eimeria species, for example, are known to be pathogenic toat least chickens, turkeys, geese, ducks, cattle, sheep, pigs, horses,rabbits, rats and mice. Toxoplasmosis of humans, puppies and lambs isassociated with Toxoplasma species, in particular, T. gondii.Cryptosporidia species infect mammals, birds and reptiles. C. muris andC. parvum in particular are known to cause gastrointestinal disease incattle, sheep and humans.

Babesiosis, associated with Babesia species, is an often fatal diseaseof domesticated animals, including cattle, horses, sheep, goats, pigs,cats and dogs. Theileria species are known to infect cattle, sheep andgoats. T. parva and T. annulata in particular, are important pathogensin cattle, the former causing African theileriosis or East Coast Fever.

One of the most intensively studied apicomplexan associated diseases ismalaria. The disease is today one of the most significant single causesof human morbidity and mortality, with estimated death rates of up to 3million and approximately 500 million infected cases per year (Butler,D. and J. Maurice, 1997).

Malaria is caused by Plasmodium species which are injected into theblood of vertebrates by female mosquito vectors. To date, fourPlasmodium species have been associated with human malaria: P.falciparum; P. vivax; P. ovate; and P. malariae. Of these, P. falciparumis believed to be the major cause worldwide. Additionally, there areknown to be at least 20 species of Plasmodium in non-human primates,including: P. cynomolgi; P. knowlesi; P. brasilianum; P. inui; P.berghei; P. yoelii; P. vinckei; and P. chabaudi.

The lifecycle of Plasmodium spp. is illustrated in Figure 0.1.

The infective stage, the sporozoites, are injected directly into thebloodstream from the salivary glands of a mosquito. These sporozoitesthen invade liver cells, within which they replicate in a processreferred to as extra-erythrocytic schizogony. At this stage, some P.vivax or P. ovale parasites will develop into hypnozoites, which remaindormant, but which, upon reactivation, may cause relapses.

After a (species-dependent) period of time, the parasites, now calledmerozoites, reinvade the circulation. The merozoites invade red bloodcells, and undergo a further phase of replication, referred to aserythrocytic schizogony. Following rupture of the infected red bloodcells, the released merozoites may in turn invade new red blood cells.This cycle of infection may be repeated many times.

The merozoites are believed to be the primary cause of malarialpathology. For example, the parasites provoke the release of cytokines,such as tumour necrosis factor, whose action is thought to beresponsible for many of the signs and symptoms of malaria. Furthermore,cerebral malaria is known to result from infected red blood cellsadhering to capillaries in the brain.

Some of the merozoites are capable of developing into the sexual stagesor gametocytes, which are taken up when a female mosquito bites again.After a period of fertilisation, ookinetes are formed and invade themosquito gut, in preparation for development into sporozoites. Thesporozoites in turn penetrate the mosquito salivary glands, in readinessfor the next bite.

Current treatments against diseases associated with apicomplexanparasites other than malaria have met with limited success. There isonly one vaccine available for this group of organisms, a liveattenuated vaccine for Toxoplasmosis that is only licenced for animaluse. Treatment with folate inhibitors and macrolides is available fortoxoplasmosis, but there is a need to develop new treatments for useduring pregnancy as the most effective treatment, sulphadiazine andpyrimethamine, is unsafe during pregnancy. Macrolides such asspiramycin, although safe are less effective and are unable to cross theblood-brain barrier, and thus is unsuitable for the treatment ofcerebral toxoplasmosis. There is currently no treatment or vaccineavailable for cryptosporidiosis.

Anti-disease strategies for malaria broadly include mosquito biteprevention, anti-parasitic drugs and prophylactic treatment.

Attempts have been made since the 1980s to use protein components of P.falciparum to develop vaccines which would stimulate the production ofprotective antibodies in a host. However, none of the vaccines testedhave provoked a strong enough immune response to be effective in thefield. SPf66, a composite vaccine targeting the blood stage, appeared toprovide some protection in field trials, but at present is not thoughtto be a suitable candidate for malarial control.

SPf66 was found to be immunogenic and to provide some level ofprotection (30-35%) in South American volunteers, but was largelyunprotective in African children who are exposed to higher levels ofinfectivity. This means that the efficacy of this vaccine is notstrain-transcending.

For any vaccine/drug to be effective, it must cross parasite-strainboundaries regardless of the geography of disease prevalence. X-rayirradiated sporozoites have been shown to be effective in a challengestudy but impractical for widespread use. A vaccine using the majorsporozoite protein, the circumsporozoite protein, CSP, did not producelong-lasting immunity as it did not induce T- cell responses. Anothervaccine based on the merozoite surface protein, MSP-1, failed to conferprotection in monkey trials. The reason why all these vaccines fail ismost probably due to a wrong choice of vaccine candidates deriveddirectly from wrong premises. Another possible problem is that theseantigens are highly polymorphic from one geographical region to theother; some are also redundant, i.e they occur in more than one copy(such as MSP-1) in the parasite. Crucially, the rationale for the choiceof vaccine candidates does not take into account the biochemistry orbiology of the cognate molecule and how it fits into the whole schema ofparasite infectivity; for example, the functions of MSP-1 or CSP whichhave been widely studied over the last 20 yrs, have not been elucidated;prior knowledge of function of a molecule is an important prerequisitefor its use in vaccine design.

Recently, attempts have been made to develop vaccines which will elicita cell-mediated immune response in the host.

One such study has aimed to produce a vaccine that would stimulate hostT-cells to destroy parasite-infected liver cells. The study has made useof a so-called “prime-boost” technique, in which the host immune systemis primed with one vaccine and boosted with another, to increase thelevels of cytotoxic T-cells. The two components of the prime-boostvaccine are: a DNA vaccine based on particular identified antigens; anda non-replicating vaccinia virus (MVA) having the gene for those sameantigens inserted in its DNA. The prime-boost vaccine has been shown toprovide protection against later malarial infection in mice. Humantrials are currently underway.

In a further example, RTSS is a viral vaccine based on sporozoiteprotein which is currently in field trials.

However, despite this apparent progress there is as yet no effectiveanti-malarial vaccine. This being so, there remains a need for newtreatments, both therapeutic and prophylactic, effective against malariaand other diseases associated with apicomplexan parasites.

SUMMARY OF THE INVENTION

Accordingly in a first aspect the present invention provides anantigenic component, for use in a vaccine capable of promoting in asubject production of an antibody specific to the antigenic component,which antibody is capable of specifically binding to the Pid proteinhaving the amino acid sequence in Seq ID No 1.

The present inventors have identified the pid (Plasmodium invasiondeterminant) locus as an invasiveness-conferring locus, occurring inapicomplexan parasites. A parasite-infected host would be expected tobear the Pid protein and antibodies to the protein, as a marker ofinfection. The Pid protein therefore provides a potential new target fortreatments against diseases associated with these parasites, includingvaccines and therapeutic agents.

Surprisingly, the newly identified Pid protein has been found to have anidentical amino acid sequence to that of the Osa (oncogenic suppressiveactivity) protein, encoded by the osa gene of the pSa plasmid (Kado, C.I. and S. M. Close, 1991; Chen, CY and C. I Kado, 1994). The osa and pidloci also have identical nucleotide sequences.

In the field of plant pathology, the pSa plasmid is known to inhibitcompletely the ability of Agrobacterium tumefaciens to incite tumours inplants. The above referenced studies reported that the osa locus aloneis sufficient for this inhibition to occur. The oncogenicity of A.tumefaciens is mediated by the transfer of a specific sector (T-DNA) ofthe bacterial Ti plasmid to the plant cell. The above studies suggestedthat the Osa protein might suppress oncogenicity by blocking thetransfer or VirE2 from bacterium to plant.

According to the first aspect of the present invention; the Pid proteinprovides a specific target for antibodies raised in a subject inresponse to a vaccine.

In general, an antibody binds to a part of a protein known as an epitopeor antigenic determinant. A protein may have more than one epitope, anddifferent epitopes on one protein may be recognised by differentantibodies. Similarly, a single antibody may be capable of binding tomore than one epitope; however the affinity of binding, and so thespecificity of the interaction, will vary.

The binding specificity of the antibodies raised in response to avaccine is determined by the antigenic component of the vaccine.Briefly, on administration of the vaccine, the antigenic component isrecognised by the host immune system, which produces antibodies capableof specifically binding to the component. In this context, bindingbetween an antigenic component and a specific cognate antibody isexpected to occur with a binding constant in the range 10⁻⁶-6 to 10⁻⁷ Mor even lower.

In, the present case, the antibodies raised in response to the vaccinemust also be capable of recognising and binding to the target Pidprotein in the infected host. In order to avoid cross-reactivity withnative host antigens., the antibodies must bind Pid specifically,typically with a binding constant in the range 1 to 10 nM and preferablybelow 1 nM.

In one embodiment, the antigenic component may comprise the Pid proteinhaving the amino acid sequence in SEQ ID No. 1, or a variant thereofwhich does not substantially affect its antigenicity. In this way,antibodies, raised to bind specifically to an epitope of the Pid proteinin the antigenic component, can bind that same epitope in the target Pidprotein.

The Pid protein with a variant sequence may be a naturally occurringvariant, or may be engineered. The variant sequence may comprise one ormore amino acid additions, substitutions or deletions compared to thesequence in SEQ ID No 1. Similarly the variant may comprise one or moremodified amino acids, provided that the variations in the amino acidsequence do not substantially affect the aritigenicity of the protein.For example, variation by conservative substitution is a possibility.Combinations of conservative substitutions are asparagine and glutamine(N or Q); valine, V, leucine, L, isoleucine, I, and methionine, M;aspartic acid and glutamic acid (D or E); lysine, K, arginine, R, andhistidine, H); alanine, A, and glycine, G; serine, S, and threonine, T;phenylalanine, F, tyrosine, Y, and tryptophan, W.

Use of a variant Pid protein may provide particular advantages. Thevariant may, for example, have improved solubility or stability, or maybe more compatible with other vaccine components. For example, a fusiontag such as thioredoxin may be linked to the protein to improvestability and/or solubility.

As explained above, a protein may comprise more than one epitope forantibody binding. Any epitope of the Pid protein may therefore becontained in only a fragment of the protein. Accordingly, the antigeniccomponent may comprise a peptide fragment of the Pid protein having theamino acid sequence in SEQ ID No 1 or a variant thereof as previouslydescribed.

Use of a peptide fragment rather than the entire protein provides asmaller antigenic component which may be more easily administered. Smallfragments may also be produced more cheaply and more easily than theintact protein.

These can be readily synthesized by synthetic chemistry or byrecombinant methods.

Preferably, the Pid protein or peptide fragment of the Pid protein inthe antigenic component is preparable from an apicomplexan parasite. Theprotein may be expressed by, and isolated from the source parasite.Alternatively, the pid locus may be isolated from a parasite andexpressed using standard cloning and expression techniques. Suitableapicomplexan parasites include those selected from the following genera:Eimeria; Isospora; Toxoplasma; Hammondia; Cystoisospora; Sarcocystis;Besnoitia; Frenkelia; Cryptosporidium; Babesia; Theileria; and, inparticular, Plasmodium.

A protein purified from the parasite source is near-to-native (havingany required post-translational modification such as myristylation orglycosylation), and therefore preferred as a source of antigeniccomponent. However, these proteins are difficult to purify in sufficientquantities. Bacterial expression is guaranteed to yield large quantitiesof recombinant protein, but this may not be post-translationallymodified. However, expression in yeast (Pichia pastoris, Saccharomycescerevisiae or Schizosaccharomyces pombe) or baculovirus/insect cellsystem, ensures that the recombinant protein is appropriately modified.

It is also however envisaged that synthetic mimics of the Pid proteinmay be constructed which are suitable for use in the antigeniccomponent.

In a vaccine aimed at eliciting an antibody response, immunogenicity ismediated by the vaccine immunogen. Accordingly, in a second aspect thepresent invention provides an immunogen comprising the antigeniccomponent coupled to an immunogenic component.

The antigenic component may itself be immunogenic so that the antigeniccomponent itself comprises the immunogenic component. However, it may bethat the antigenic component is, for example, too small to beimmunogenic to the host. In that case, it may be necessary to couple theantigenic component to a suitable carrier. To be effective, therefore,the isolated antigen preferably stimulates the host immune system in amanner and at a level similar to that elicited during biologicalinfection. To enhance antigen presentation and immunogenicity, it may becoupled to haptens such as bovine serum albumin and keyhole limpethaemocyanin; viral particles or dendrimers. It may also be possible toengineer attenuated Salmonella strains to carry vaccines to be deliveredorally as live vaccines. Salmonella is appropriate for this purposebecause it induces both high antibody and cell-mediated immuneresponses.

In a further aspect, the present invention provides a vaccine comprisingan immunogen and an adjuvant, which enhances the antibody response.Freund's complete adjuvant and Freund's incomplete adjuvant, forexample, are suitable for use in non-human vaccines. Aluminium hydroxideand aluminium phosphate are adjuvants authorised for human use. Possiblefurther adjuvants include liposomes, BCG, lipopolysaccharides, muramyldipeptide derivatives, squalene, non-ionic hydrophobic block copolymersurfactants such as polyoxypropylene and polyoxyethylene copolymers,pluronic polyols, ethylene-vinyl acetate, cyclodextrins and polysialicacid.

In a further aspect the present invention provides a vaccine comprisinga polynucleic acid, which encodes the antigenic component describedabove. The polynucleic acid may comprise, for example, DNA, RNA or asynthetic nucleic acid.

In one embodiment, the polynucleic, acid comprises the sequence in SEQID No 2.

Polynucleic acid vaccines are typically aimed at eliciting acell-mediated immune response in a subject. A key feature of this typeof vaccine is that the antigenic component encoded by the polynucleicacid of the vaccine is expressed in and displayed on the surface of acell within the subject. These vaccines may be of particular use againstdiseases associated with cell-invasive parasites, since they mimic thenatural situation where the antigen is intracellular.

The polynucleic acid of the present vaccine may further comprisesequence for efficient expression of the antigenic component. Forexample, such sequence may comprise a promoter sequence or encode asecretion signal.

Advantageously, the present vaccine also comprises a delivery means fordelivery of the polynucleic acid to a subject. The vaccine mayadditionally comprise an adjuvant for enhancing the cell-mediated immuneresponse in the subject.

The present polynucleic acid vaccine preferably takes either of two mainforms: a naked vaccine or a live vaccine.

In the case of a naked vaccine, the polynucleic acid is administered toa subject, and by one of a number of alternative means, is delivered toa target host cell. The antigenic component encoded by the polynucleicacid is then expressed by the host cell.

The polynucleic acid of a naked vaccine may, for example, comprise aplasmid, bearing a eukaryotic promoter to direct efficient expression ofthe antigenic component in a target host cell. The promoter may beconstitutive, for example, the generic CMV promoter or SV40 promoter.Alternatively, the promoter may be tissue specific. In one embodimentthe promoter is a muscle specific promoter such as the MyoD, myosin ormyogenin promoters. The significance of a muscle-specific promoter isthat a polynucleic acid vaccine delivered by intramuscular injection canbe expressed directly by muscle cells.

The liver may also be targeted for expression of antigens; aliver-specific promoter such as the albumin promoter may be used incombination with a secretory signal tagged onto the antigen open readingframe for expression and secretion.

The polynucleic acid may additionally comprise sequence encoding asecretion signal for the antigenic component, to ensure that duringexpression the component is secreted to the outer surface of the hostcell. For example, the secretion signal may comprise the malE signal forbacterial expression; the honeybee melittin signal for baculovirusexpression in insect cells (Sf9; Sf21); the α-factor for expression inyeast and the Igκ signal for expression in mammalian cells.

The polynucleic acid of the naked vaccine may further compriseimmunostimulatory sequences which provide a suitable adjuvant. Forexample, unmethylated CpG sequences may be used for this purpose. CpGimmunostimulatory sequences may also be combined with one or more otheradjuvants indicated above such as complete or incomplete Freund'sadjuvant.

For delivery to a subject, the polynucleic acid of the naked vaccine maybe complexed with for example, liposomal vesicles or viral particles. Inone embodiment, the vaccine is delivered by injection through the skinor muscle. In a further embodiment, the vaccine is delivered bynebulisation. Liposomes and viral particles act as carriers. Noparticular cell types are targeted except when tissue-specificexpression is desired wherein the requisite promoter will be included onthe delivered DNA sequence.

In the case of a live vaccine, the polynucleic acid is first transformedinto a suitable strain of bacteria, so that the bacterial cells expressthe antigenic component on their cell surface. The expressing bacterialstrain is then administered to the subject, so that the bacteria providethe required delivery means.

Bacterial strains suitable for this purpose include attenuatedaro/auxotrophic mutants of Salmonella, Listeria and Corynebacterium,pseudotuberculosis. Viral vectors such as attenuated herpes simplex, BCGand adenoviruses can also be used.

The polynucleic acid of a live vaccine may comprise an expressionvector, bearing a prokaryotic promoter to direct efficient expression ofthe antigenic component in the bacterium. Suitable promoters forbacterial expression include the tac, trc, BAD, T7 and P_(L)/trppromoters. The polynucleic acid may also encode a secretion signal,directing secretion of the component from the bacterial cell, therebyexposing the component to the host immune system. Examples of secretionsignals include malE, ompT, pelB and bacteriophage fd gene III proteinsignal.

In a further embodiment the polynucleic acid encoding the antigeniccomponent may be integrated into the bacterial chromosome. Integrationwould be expected to provide improved stability and increased expressionlevels.

Expression of the antigen will typically be driven by any one of theabove generic promoters. Alternatively, it may be possible to use, forexample, a Salmonella gene promoter. The secretion signal will be anintegral part of any targeting recombinant vaccine vector, and thereforewill be stably integrated into the bacterial chromosome.

In the case of the live vaccine, the use of an adjuvant may be optimalwhere, for example, Salmonella is used because Salmonella is able toinduce secretory, humoral and cellular immunity.

Preferably, the vaccines described above are suitable for use in a humansubject. These vaccines will, for example, comprise an adjuvant suitablefor use in humans, such as those described above. Typically, thesevaccines will be non-pyrogenic, non-inflammatory and non-necreotizing,as well as being protective against biological infection.

In one embodiment, the present vaccine is suitable for use against humanmalaria caused by P. falciparum, P. ovale, P. vivax, or P. malariae.

Without wishing to be bound by theory, the present vaccine may targetPid at one or more of the life-cycle stages of an apicomplexan parasite.In the case of a Plasmodium parasite, the vaccine may, for example,target a lifecycle stage which is invasive to mammalian liver cells orred blood cells. The target life-cycle stage may be the sporozoiteswhich invade the liver; the merozoites which invade red blood cells orthe ookinetes, which invade the mosquito gut wall during or after ablood meal to complete development to sporozoites.

In addition to providing a new target for a vaccine, the Pid proteinalso provides a basis for new therapies against infectious disease, suchas apicomplexan-associated disease.

Accordingly, in a further aspect the present invention provides atherapeutic agent comprising a component which component is capable ofcompeting with a protein having the amino acid sequence in Seq ID No. 1in a specific binding assay.

It is envisaged that the Pid protein has a critical role in cellinvasion by, for example, apicomplexan parasites. Without wishing to bebound by theory this is likely to occur by interaction of Pid with areceptor. A component which is capable of competing with Pid in an invitro specific binding assay is likely to be capable of competing withPid in vivo for receptor binding. The component can therefore beincorporated into a therapeutic agent aimed at blocking Pid-receptorinteraction.

In one embodiment, Pid can be used in combinatorial phage displayselection from a pool of random peptides without prior knowledge of itstarget receptor(s) or interactors. A library of random peptides of up to15 amino acids may be constructed and displayed on a phage surface.Recombinant Pid protein is then immobilized on Petri dishes, blockedwith BSA to occlude non-specific sites. The phage-encoded peptides arethen incubated with Pid. After this step, non- specifically boundpeptides/phage are washed off and specific phage are eluted, amplifiedin permissive bacterial hosts and the whole panning cycle is repeated.After 4-5 selections, the peptide sequences are determined; the affinityof binding may then be further optimized by site-directed mutagenesis.These peptides are then synthesized and used in binding assays. Highaffinity binding may be defined as those peptides with a dissociationconstant in the 1-10 nM range. IN₅₀ may be determined by competitiveELISA. Typically, a value of 10⁻⁶ to 10⁻⁷ is deemed competitive binding.

In one embodiment, It is envisaged that a mimic of the Pid protein maybe produced and incorporated into a therapeutic agent. Alternatively,the receptor for Pid may be identified and an inhibitor of thePid-receptor interaction used as a component of the therapeutic agent.

Antibodies to a Pid receptor may also be therapeutic since they mayblock interaction with Pid, assuming that receptor antibody epitopes andbinding sites for Pid are the same.

In a further aspect the invention provides a protein comprising theamino acid sequence in SEQ ID No 1 or a fragment thereof for use inmedicine. The invention additionally provides a polynucleic acidencoding the protein or a fragment of the protein, for use in medicine.

The vaccine and the therapeutic agent, both according to the presentinvention, are suitable for use in methods of treatment againstinfectious diseases such as those caused by apicomplexan parasites.

This aspect of the present invention therefore advantageously providesthe use of the above antigenic component for the manufacture of amedicament effective against a disease caused by an apicomplexanparasite.

According to the above, manufacture of a medicament effective againstsuch a disease may comprise the use of a protein comprising the aminoacid sequence in SEQ ID No 1 or a peptide fragment thereof, and/or theuse of a polynucleic acid encoding such a protein or peptide fragment.

Diseases which may suitably be treated according to the presentinvention include those caused by apicomplexan parasites of thefollowing genera: Eimeria; Isospora; Toxoplasma; Hammondia;Cystoisospora; Sarcocystis; Besnoitia; Frenkelia; Cryptosporidium;Plasmodium; Babesia; and Theileria.

Preferably, the disease is one selected from the following: malaria;coccidiosis; theileriosis; cryptosporidiosis; isosporiasis;blastocystosis; babesiosis; anaplasmosis; sarcosporidiosis;toxoplasmosis; and sarcosystosis.

In particular, the invention provides a means for preventing andtreating malaria disease, associated with the Plasmodium parasite. Humanmalaria, associated with for example, P. falciparum, P. vivax, P. ovaleand P. malariae, is a particularly important target.

As noted above, the Pid protein provides a convenient marker ofinfection by an organism bearing the pid locus, such as an apicomplexanparasite. Accordingly, in one aspect the present invention provides adiagnostic agent comprising an antibody, which antibody is capable ofspecifically binding to the Pid protein having the amino acid sequencein SEQ ID No 1.

In this context, the antibody is typically capable of binding to the Pidprotein with a binding constant in the range from 10⁻⁶ to 10⁻⁷, Thediagnostic agent preferably comprises a means for detecting antibody-Pidcomplexes. The antibody may, for example, be labelled using standardtechniques with a fluorophore, a radiolabel, a marker enzyme or aligand. Suitable fluorophores include fluorescein, rhodamine, Cy3, TRITCand phycoerythrin. Suitable marker enzymes include alkaline phosphatase,beta galactosidase and horse radish peroxidase. Suitable ligands includebiotin and digoxigenin (DIG).

In a further aspect the invention provides the use of the diagnosticagent in a method of diagnosing disease caused by an apicomplexanparasite. The diagnostic agent may for example be used in vitro todetect the presence of the Pid protein in a sample taken from a subject.

An in vitro diagnostic test of infection based on the detection of pidantibodies in patient serum/plasma may involve ELISA or EIA which may becomplemented with immunofluorescence microscopy. Any diagnostic agentinvolving pid should include positive controls (purified pid protein andthe cognate reactive antibodies) as well substrates for the relevantlabel [eg. p-nitrophenyl phosphate (NPP) for alkaline phosphate(AP)-labelled secondary antibodies].

Infection may also be identified by detecting antibodies to a Pidprotein in the serum of a subject. Accordingly in a further aspect thepresent invention provides a diagnostic agent comprising an antigeniccomponent as described above.

The antigenic component will bind to any Pid antibodies present in thesubject sera; binding can be detected by one of a number of standardlabelling techniques.

The present invention further provides the use of the diagnostic agentin a method of diagnosing disease caused by an apicomplexan parasite.

In one embodiment, the antigenic component of the diagnostic agent maybe immobilised onto ELISA plates, and incubated with subject sera.Antibodies to Pid in the sera may then be detected by antigen capture,with monoclonal antibodies to Pid being used as a positive control.

Other labeling and detection methods may include chromogenic methods[e.g. AP-NPP or nitrotetrazolium blue and 5-bromo-4-chloro-3-indolylphosphate (BCIP)], fluorogenic (e.g. biotin and fluorescein-labelledstreptavidin) or luminescence (e.g AP and CDP-Star). While ELISA or EIAmethods may be used, it may also be possible to perform Western blots orfluorescence in situ hybridization. In some cases, detection may beenhanced by in situ polymerase chain reaction using fluorogenicnucleotides.

The immobilised antigenic component may be provided in a spot test ordip-stick method for field diagnosis. Hapten- conjugated (FITC, alkalinephosphatase etc) monoclonal antibodies to Pid will then be incubatedwith parasites and visualised directly by fluorescence microscopy.

In accordance with the above, the present invention provides anantibody, which is capable of specifically binding to the Pid proteinhaving the amino acid sequence in SEQ ID No 1 for use in medicine.

The invention also provides the use of such an antibody for themanufacture of a diagnostic agent for diagnosis of a disease caused byan apicomplexan parasite.

In a further aspect, the invention provides the use of an antigeniccomponent as described above, for the manufacture of a diagnostic agentfor diagnosis of a disease caused by an apicomplexan parasite.

It will be appreciated that to manufacture the diagnostic agentsdescribed above may comprise the use of a protein comprising the aminoacid sequence in SEQ ID No 1 or a fragment thereof, or the use of apolynucleic acid encoding the protein or peptide fragment.

A diagnostic agent may be provided in a kit typicaly comprising positivecontrols (purified pid protein and the cognate reactive antibodies) aswell, substrates for the relevant label [eg. p-nitrophenyl phosphate(NPP) for alkaline phosphate (AP)-labelled secondary antibodies]. Adetailed and fully referenced instruction manual and calibrationinformation would normally be an integral part of the kit.

In a further aspect there is provided an in vitro method for diagnosingapicomplexan infection in a subject, which comprises:

-   (i) obtaining from the subject a nucleic acid containing sample; and-   (ii) testing the sample for the presence of nucleic acid sequence    characteristic of Pid. The nucleic acid sequence characteristic of    Pid may be all or a part of the sequence encoding the Pid protein or    may be nucleic acid sequence upstream or downstream of the Pid    coding sequence. Conveniently, the nucleic acid containing sample is    subjected to a step of amplification, such as by PCR, which is    preferably specific to the Pid nucleic acid sequence. This may be    achieved using appropriate primers, such as any of those set out in    FIG. 5. Other primers unique to the target nucleic sequence may also    be used.

Infection by any of the apicomplexans described above may be detectedusing this method. Where the infection is in humans, detection ofplasmodium infection is particularly important in diagnosing malaria.This may be achieved using red blood cells as the sample source ofnucleic acid.

The apicomplexan associated diseases which may be diagnosed according tothe present invention include those described above.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in more detail by way of exampleonly with reference to the accompanying drawings in which

FIG. 1 illustrates the lifecycle of a Plasmodium parasite;

FIGS. 2 a and 2 b shows the results of an immunofluorescence invasionassay on E. coli strains transformed with cosmid clones of P. yoeli DNA;

FIG. 2 c shows transmission electron micrographs of COS-7 cells whichhave been invaded by E. coli strains transformed with ‘Inv’ cosmidclones of P. yoeli DNA;

FIG. 3 provides a bar chart displaying the results of plate scoring ofwild type cosmid clones (Invcos11WT and Invcos18WT) andTn1000γδ-inserted clones (TM1 and TM5), rescued from COS-7 cellsfollowing invasion assays;

FIG. 4 a shows a nucleotide sequence including the PID nucleotidesequence (SEQ ID:NO. 2) and associated amino acid sequence (SEQID:NO. 1) according to the invention;

FIG. 4 b shows a schematic representation of PID and adjacent sequences;and

FIG. 5 shows a part of the Pid nucleotide sequence identifying primersites for diagnostic assays;

FIG. 6 shows results of PCR amplification of Pid from patient samples;and

FIG. 7 shows Pid sequences amplified from patients.

DETAILED DESCRIPTION OF THE INVENTION Examples

Isolation of the pid locus from Plasmodium yoeli.

As described above, the lifecycle of Plasmodium sp. involves threeinvasive stages (FIG. 1): the ookinete; the sporozoite and themerozoite. Each stage has a different target cell preference. In thehost, for example, sporozoites invade liver cells preferentially,whereas merozoites invade red blood cells.

In general terms there are three major steps in Plasmodium invasion:recognition, attachment and entry. The factor or factors which areinvolved in the invasive process have not been conclusively identified.A number of possible parasite specific ligands have been implicated incell entry, and various cellular structures suggested as putativereceptors. However these studies have focused on binding to the cellrather than on actual cell entry (Sim, B. K. L et al, 1994; Horuk, R etal, 1993; Breton, C. B et al, 1992; and Hadley, T J et al, 1986).

Using a reconstitution assay (Isberg, R. R. and S. Falkow, 1985), thepresent inventors have isolated from P. yoeli (a murine malariaparasite), a locus that surprisingly is both necessary and sufficientfor invasion of epithelial cells by E. coli K12.

Initially, cosmid clones of segments of the P. yoeli genome weretransduced into E. coli. The clones were screened for the ability tobind to and invade cultured COS-7 cells. In the screening process,primary validation of invasion or internalisation was based ongentamicin-killing; gentamicin permeates cells very poorly and cantherefore be used to eliminate cell-surface bound bacteria.

On the basis of the screening method, several invasive clones (‘Inv’)were isolated and found to cross-hybridise.

Restriction mapping of the clones further showed that they overlappedwith each other.

In order to confirm that the identified clones were indeed invasive,further invasion assays were carried out using E. colii K12 strainstransformed with the cosmid clones.

The assays were carried out as above but the bacteria were tracked byindirect immunofluorescence using a primary polyclonal antibody specificto E. coli K12. Punctate fluorescence of invading bacteria was observed.No invasion was observed when the assay was carried out with controlstrains: untransformed E. coli or bacteria transformed with plasmid orcosmid vector.

To establish that the bacterial cells were internalised rather thanextracellular, the invaded cells were counter-stained withTRITC-labelled phalloidin. Actin polymerisation could be observed atfoci of cell entry and invading bacteria colocalised with the nucleationof polymerised actin filaments.

To provide further proof of cell entry, transmission electron microscopywas performed on the invaded cultured cells. Bacteria were found withinmembrane-bound vacuoles.

In order to investigate further the role of this locus in cell entry,one invasion-proficient clone, Invcos18, was subjected to transposonmutagenesis with Tn1000γδ (Morris, G. E et al, 1995).

97% of the transposon mutants were severely impaired for cell invasion,and were unable to complement E. coli in cell entry.

Sequence analysis of six non-complementing clones (with cfu≦1) showedtransposon insertion at the same site in all six cases, suggesting thatthe inserted locus was indispensable for invasion. FIG. 4A shows thenucleotide sequence of the region, with the location of the transposonends marked in sequence.

Transposon insertion sites were located which intercept a putative openreading frame of about 567 nucleotides. The sequence of the pid locus isdesignated SEQ ID no 2 and runs from nucleotide 607 in FIG. 4A tonucleotide 1172.

The predicted amino acid sequence of the pid translation product ispresented in FIG. 4B and is designated SEQ ID No.1. This is also shownin FIG. 4A. The DNA ssequence upstream of Pid is designated SEQ ID NO:3and encodes an amino acid sequence from ORF3, designated SEQ ID No.4.The sequence downstream of Pid is designated SEQ ID NO:5.

The BLAST programme was used to search the GenBank, EMBL, DDBJ and PDBdatabases for sequences homologous to the pid or Pid sequences.

The pid locus was found to be a pathogenicity island characterised by anunusually high GC content (55%) compared to parasite chromosomal DNA.DNA sequences contiguous with the invasion locus are more AT rich andshow homology (about 38%) to the transmissible TraC/primase locus, whichis required for conjugal transfer of the broad host range plasmids, IncNand IncW (Valentine, C. R. I. and C. I. Kado, 1989). Anatol, A.Belogurov et al, Antirestriction protein Ard (Type C) encoded by IncWplasmid pSa has a high similarity to the protein transport domain ofTrac1 primase of promiscuous plasmid RP4. Journal of Molecular Biology,(2000), 296: 969-977.

Without wishing to be bound by theory, this suggests the presence ofintegrated cryptic plasmid DNA sequences in the parasite genome. It ispossible that the pid locus has been acquired by horizontal transfer ofa primordial gene derived from a pathogenic bacterium or bacteriophage.

In the homology searches, the Pid protein amino acid sequence was foundto be identical to that of the Osa protein, encoded by the osa gene onthe Shigella flexneri virulence plasmid pSa. The pid and osa nucleotidesequences are identical.

The Pid protein also shows homology to the product of the internalin Blocus (InlB) in the inlAB operon of Listeria. monocytogenes, which hasbeen shown to be indispensable for invasion (Gaillard, J. L. et al,1991; Parida, S. K. et al, 1998).

The inventors have also identified a putative CDC42/Rac- interactivebinding (CRIB) motif:

-   -   PVLSRDEASAVMLAEHVGVA

in the Pid protein sequence. This motif is designated SEQ ID No65. Themotif is associated with small GTPase- effector proteins such as N-WASP,Ste20 and MSE55. An alignment of the sequences of Pid and other CRIBproteins is shown in FIG. 4B. The alignment was produced using the BLASTprogramme. The putative CRIB domain in pid does not align with theinternalin B sequence. The homology of the latter and of the RhoGTPase-effector proteins was identified by a BLASTP search. TheAccession numbers for pid homologues are as follows: Internalin B. Acc #AF 121032 Cdc42 effector protein (MSE55) Acc # XM_001058.1 STE20 Acc #L04655 WASP/human Acc # NM 000377 p65^(PAK) -d/rat Acc # NM 017198

Apart from internalin, all the rest have CRIB domains.

WASP, Ste20 and MSE55 proteins are known to have an effect on actincytoskeletal reorganisation (Burbelo, P. D. et al, 1999; Hall, A et al,1998; Burbelo, P. D et al, 1998). In Salmonella typhimurium, forexample, an interaction of N- WASP and SopE with the Rho GTPases CDC42and Rac1, is required for invasion of non-phagocytic cells (Hardt, W. Det al, 1998; Chen, L. M. et al 1996; Susuki, T et al, 1998; Masol, P etal, 1998). Thus, Pid may be a guanine nucleotide exchange factor or aGTPase-activating protein.

Isolation and expression of Pid.

The pid gene may be isolated from a suitable parasite using standardcloning techniques, and the sequence information in SEQ ID No1 and No2.Fragments of the gene may be obtained using standard methods.

The pid gene or fragments of the gene may be expressed, and the proteinproducts purified using conventional methods. For details of the abovemethods the reader is referred to Riggs, P., in Ausubel, F. M. et al(eds) Current Protocols in Molecular Biology John Wiley & Sons, Inc.,New York, pp 16.6.1-16.6.14 (1994)

Vaccine Production.

Antibody-mediated Vaccine

Once an antigenic component has been obtained, a vaccine comprising theantigenic component may be produced by conventional means. The reader isdirected towards the following references:

-   Jane L. Holley, Mike Elmore, Margaret Mauchline, Nigel Minton and    Richard W. Titball.Cloning, expression and evaluation of a    recombinant sub-unit vaccine against Clostridium botulinum type. F    toxin [Abstract] [Full text] Vaccine 19 (2-3) 288-297 (2000)-   Michael Theisen et al. Identification of a major B-cell epitope of    the Plasmodium falciparum glutamate-rich protein (GLURP), targeted    by human antibodies mediating parasite killing; Vaccine 19 (2-3)    204-212 (2000)-   J H Tian, S Kumar, D C Kaslow, and L H Miller Comparison of    protection induced by immunization with recombinant proteins from    different regions of merozoite surface protein 1 of Plasmodium    yoelii Infect. Immun. 1997 65: 3032-3036.-   Bittle, J. L., Houghten, R. A., Alexander, H., Shinnick, T. M.,    Sutcliffe, J. G., Lerner, R. A., Rowlands, D. J. and Brown, F. 1982.    Protection against foot-and-mouth disease by immunization with a    chemically synthesized peptide predicted from the viral nucleotide    sequence. Nature 298:30-33.-   Harlow, E. & Lane, D. 1988. Antibodies: A laboratory manual, Cold    Spring Harbour Laboratory Press, Cold Spring Harbour, New York.-   Holder, A. A. and Freeman, R. R. 1981. Immunization against    bloodstage-rodent malaria using purified parasite antigens, Nature,    294:361-364.-   Hollingdale, M. R., Nardin, E. H., Tharavanij, S., Schwartz, A. L.    and Nussenzweig, R. S. 1984. Inhibition of entry of sporozoites of    Plasmodium falciparum and Plasmodium vivax sporozoites into cultured    cells; an in vitro assay of protective antibodies. J. Immunol.    132:909-913.-   Kaslow, D. C., Quakyi, I. A., Syin, C., Raum, M. G., Keister, D. B.,    Coligan, J. E., McCutchan, T. F. and Miller, L. H. 1988. A vaccine    candidate from the sexual stage of human malaria that contains    EGF-like domains. Nature 333:74-76.

The vaccine may be administered to a test subject and the antibodiesraised in response to the vaccine tested for specific binding to Pid,both using standard methods. Suitable methods are described for examplein the following references: J H Tian, S Kumar, D C Kaslow, and L HMiller Comparison of protection induced by immunization with recombinantproteins from different regions of merozoite surface protein 1 ofPlasmodium yoelii .Infect. Immun. 1997 65: 3032-3036.

-   Harlow, E. & Lane, D. 1988. Antibodies: A laboratory manual, Cold    Spring Harbour Laboratory Press, Cold Spring Harbour, New York.-   Holder, A. A. and Freeman, R. R. 1981. Immunization against    bloodstage-rodent malaria using purified parasite antigens, Nature,    294:361-364.-   Hollingdale, M. R., Nardin, E. H., Tharavanij, S., Schwartz, A. L.    and Nussenzweig, R. S. 1984. Inhibition of entry of sporozoites of    Plasmodium falciparum and Plasmodium vivax sporozoites into cultured    cells; an in vitro assay of protective antibodies. J. Immunol.    132:909-913.-   Kaslow, D. C., Quakyi, I. A., Syin, C., Raum, M. G., Keister, D. B.,    Coligan, J. E., McCutchan, T. F. and Miller, L. H. 1988. A vaccine    candidate from the sexual stage of human malaria that contains    EGF-like domains. Nature 333:74-76.-   Armando Reyna-Bello, Axel Cloeckaert, Nieves Vizcaííno, Mary I.    Gonzatti, Pedro M. Aso, Gééraird Dubray, and Michel S. Zygmunt    Evaluation of an Enzyme-Linked Immunosorbent Assay Using Recombinant    Major Surface Protein 5 for Serological Diagnosis of Bovine    Anapiasmosis in Venezuela Clin. Diagn. Lab. Immunol. 1998 5:    259-262.-   M Theisen, J Vuust, A Gottschau, S Jepsen, and B Hogh Antigenicity    and immunogenicity of recombinant glutamate- rich protein of    Plasmodium falciparum expressed in Escherichia coli Clin. Diagn.    Lab. Immunol. 1995 2: 30-34.

Similarly, the vaccine may be tested for effectiveness against diseasesuch as those caused by apicomplexan parasites, using standardlaboratory protocols. These may be, found, for example, in the followingreferences: J H Tian, S Kumar, D C Kaslow, and L H Miller Comparison ofprotection induced by immunization with recombinant proteins fromdifferent regions of merozoite surface protein 1 of Plasmodium yoeliiInfect. Immun. 1997 65: 3032-3036.

-   Harlow, E. & Lane, D. 1988. Antibodies: A laboratory manual, Cold    Spring Harbour Laboratory Press, Cold Spring Harbour, New York.-   Holder, A. A. and Freeman, R. R. 1981. Immunization against    bloodstage-rodent malaria using purified parasite antigens, Nature,    294:361-364.-   Hollingdale, M. R., Nardin, E.-H., Tharavanij, S., Schwartz, A. L.    and Nussenzweig, R. S. 1984. Inhibition of entry of sporozoites of    Plasmodium falciparum and Plasmodium vivax sporozoites into cultured    cells; an in vitro assay of protective antibodies. J. Immunol.    132:909-913.-   Kaslow, D. C., Quakyi, I. A., Syin, C., Raum, M. G., Keister, D. B.,    Coligan, J. E., McCutchan, T. F. and Miller, L. H. 1988. A vaccine    candidate from the sexual stage of human malaria that contains    EGF-like domains. Nature 333:74-76.-   Armando Reyna-Bello, Axel Cloeckaert, Nieves Vizcaííno, Mary I.    Gonzatti, Pedro M. Aso, Géérard Dubray, and Michel S. Zygmunt    Evaluation of an Enzyme-Linked Immunosorbent Assay Using Recombinant    Major Surface Protein 5 for Serological Diagnosis of Bovine    Anaplasmosis in Venezuela Clin. Diagn. Lab. Immunol. 1998 5:    259-262.-   M Theisen, J Vuust, A Gottschau, S Jepsen, and B Hogh Antigenicity    and immunogenicity of recombinant glutamate- rich protein of    Plasmodium falciparum expressed in Escherichia coli Clin. Diagn.    Lab. Immunol. 1995 2: 30-34.    Polynucleic Acid Vaccine

Once a suitable polynucleic acid has been identified, the polynucleicacid may be incorporated, in a vaccine using conventional methods. Inthis regard the reader is directed following documents: (Jones, T. R. etal, 1.999; Chatfield, S. N. et al, 1989;) Ricardo S. Corral and PatriciaB. Petray CpG DNA as a Th1-promoting adjuvant in immunization againstTrypanosoma cruzi Vaccine 19 (2-3) 234-242 (2000)

-   K. D. Song et. A DNA vaccine encoding a conserved Eimeria protein    induces protective immunity against live Eimeria acervulina    challenge Vaccine 19 (2-3) 243-252 (2000)-   Rong Xiang, Holger N. Lode, Ta-Hsiang Chao, J. Michael Ruehlmann,    Carrie S. Dolman, Fernando Rodriguez, J. Lindsay Whitton, Willem W.    Overwijk, Nicholas P. Restifo, and Ralph    A. Reisfeid An autologous oral DNA vaccine protects against murine    melanoma PNAS 97: 5492-5497-   Martha Sedegah, Trevor R. Jones, Manjit Kaur, Richard Hedstrom,    Peter Hobart, John A. Tine, and Stephen L. Hoffman Boosting with    recombinant vaccinia increases immunogenicity and protective    efficacy of malaria DNA vaccine PNAS 95: 7648-7653.-   Donald L. Lodmell, Nancy B. Ray and Larry C. Ewalt Gene gun    particle-mediated vaccination with plasmid DNA confers protective    immunity against rabies virus infection, Vaccine 16 (2-3) 115-118    (1998)-   Cynthia L. Brazolot Millan, Risini Weeratna, Arthur M. Krieg,    Claire-Anne Siegrist, and Heather L. Davis CpG DNA can induce strong    Th1 humoral and cell-mediated immune responses against hepatitis B    surface antigen in young mice PNAS 95: 15553-15558.-   Wendy C. Brown, D. Mark Estes, Sue Ellen Chantler, Kimberly A.    Kegerreis, and Carlos E. Suarez DNA and a CpG Oligonucleotide    Derived from Babesia bovis Are Mitogenic for Bovine B Cells Infect.    Immun. 1998 66: 5423-5432.-   Zina Moldoveanu, Laurie Love-Homan, Wen Qiang Huang and Arthur M.    Krieg. CpG DNA, a novel immune enhancer for systemic and mucosal    immunization with influenza virus Vaccine, 16(11-12)1216-1224

The polynucleic acid vaccine may be administered to a subject and testedfor therapeutic efficacy using standard protocols, such as those foundin the following references:

-   J H Tian, S Kurrar, D C Kaslow, and T H Miller Comparison of    protection induced by immunization with recombinant proteins from    different regions of merozoite surface protein 1 of Plasmodium    yoelii .Infect. Immun. 1997-65: 3032-3036.-   Bittle, J. L., Houghten, R. A., Alexander, H., Shinnick, T. M.,    Sutcliffe, J. G., Lerner, R. A., Rowlands, D. J. and Brown, F. 1982.    Protection against foot-and-mouth disease by immunization with a    chemically synthesized peptide predicted from the viral nucleotide    sequence. Nature 298:30-33.-   Harlow, E. & Lane, D. 1988. Antibodies: A laboratory manual, Cold    Spring Harbour Laboratory Press, Cold Spring Harbour, New York.-   Holder, A. A. and Freeman, R. R. 1981. Immunization against    bloodstage-rodent malaria using purified parasite antigens, Nature,    294:361-364.-   Hollingdale, M. R., Nardin, E. H., Tharavanij, S., Schwartz, A. L.    and Nussenzweig, R. S. 1984. Inhibition of entry of sporozoites of    Plasmodium falciparum and Plasmodium vivax sporozoites into cultured    cells; an in vitro assay of protective antibodies. J. Immunol.    132:909-913.-   Kaslow, D. C., Quakyi, I. A., Syin, C., Raum, M. G., Keister, D. B.,    Coligan, J. E., McCutchan, T. F. and Miller, L. H. 1988. A vaccine    candidate from the sexual stage of human malaria that contains    EGF-like domains. Nature 333:74-76.-   M Theisen, J Vuust, A Gottschau, S Jepsen, and B Hogh Antigenicity    and immunogenicity of recombinant glutamate- rich protein of    Plasmodium falciparum expressed in Escherichia coli Clin. Diagn.    Lab. Immunol. 1995 2: 30-34.    Design and production of therapeutic agent

Once a pid gene is isolated, and the Pid protein expressed and purified,a component for a therapeutic agent may be obtained by conventionalmethods.

The Pid protein may, for example, be crystallised and the 3-D structuredetermined, by methods such as those set out in Alex M. Aronov, StephenSuresh, Frederick S. Buckner, Wesley C. Van Voorhis, Christophe L. M. J.Verlinde, Fred R. Opperdoes, Wim G. J. Hol, and Michael H. GelbStructure- based design of submicromolar, biologically active inhibitorsof trypanosomatid glyceraldehyde-3-phosphate dehydrogenase PNAS 96:4273-4278.

This structure may be used as a template to design Pid mimics bycombinatorial chemistry; the reader is directed towards the followingdocuments: (Aronov, A. M. et al, 1999; ‘Combinatorial chemistry’ (1996)Methods in Enzymology vol 267 ed. John N Abelson; Academic Press Inc.New York;) Kirk McMillan, Marc Adler, Douglas S. Auld, John J. Baldwin,Eric Blasko, Leslie J. Browne, Daniel Chelsky, David Davey, Ronald E.Dolle, Keith A. Eagen, Shawn Erickson, Richard I. Feldman, Charles B.Glaser, Cornell Mallari, Michael M. Morrissey, Michael H. J. Ohlmeyer,Gonghua Pan, John F. Parkinson, Gary B. Phillips, Mark A. Polokoff,Nolan H. Sigal, Ronald Vergona, Marc Whitlow, Tish A. Young, and JamesJ. Devlin Allosteric inhibitors of inducible nitric oxide synthasedimerization discovered via combinatorial chemistry PNAS 97: 1506-1511.

-   Dustin J. Maly, Ingrid C. Choong, and Jonathan A. Ellman    Combinatorial target-guided ligand assembly: Identification of    potent subtype-selective c-Src inhibitors PNAS 97: 2419-2424.-   R A P Lutzke, N A Eppens, P A Weber, R A Houghten, and R H A    Plasterk Identification of a Hexapeptide Inhibitor of the Human    Immunodeficiency Virus Integrase Protein by Using a Combinatorial    Chemical Library PNAS 92: 11456-11460-   N F Sepetov, V Krchnak, M Stankova, S Wade, K S Lam, and M Lebl    Library of Libraries: Approach to Synthetic Combinatorial Library    Design and Screening of “Pharmacophore” Motifs PNAS 92: 5426-5430-   J J Burbaum, M H J Ohlmeyer, J C Reader, I. Henderson, L W Dillard,    G Li, T L Randle, N H Sigal, D Chelsky, and J J Baldwin A Paradigm    for Drug Discovery Employing Encoded Combinatorial Libraries PNAS    92: 6027-6031-   D. T. O'Hagan et al. Microparticles in MF59, a potent adjuvant    combination for a recombinant protein vaccine against HIV-1 Vaccine    18(17):1793-1801 (2000)-   Jing Huang and Stuart L. Schreiber A yeast genetic system for    selecting small molecule inhibitors of protein-protein interactions    in nanodroplets PNAS 94: 13396-13401.-   Jay Parrish, Helen Metters, Lin Chen, and Ding Xue Demonstration of    the in vivo interaction of key cell death regulators by    structure-based design of second-site suppressors PNAS 97:    11916-11921

The mimics may be tested for competitive binding with Pid in a specificbinding assay according to the methods set out in Harlow, E. & Lane, D.1988. Antibodies: A laboratory manual, Cold Spring Harbour LaboratoryPress, Cold Spring Harbour, New York.

The pharmacophores may be assessed by QSAR (quantitativestructure-activity relationships); their toxicity may be tested in vivoand their efficacy as therapeutic agents assessed by the level ofprotection they confer from parasite infection. For suitable protocols,the reader is directed towards: Alex M. Aronov, Stephen Suresh,Frederick S. Buckner, Wesley C. Van Voorhis, Christophe L. M. J.Verlinde, Fred R. Opperdoes, Wim G. J. Hol, and Michael H. GelbStructure-based design of submicromolar, biologically active inhibitorsof trypanosomatid glyceraldehyde-3-phosphate dehydrogenase PNAS 96:4273-4278.

-   NF Sepetov, V Krchnak, M Stankova, S Wade, K S Lam, and M Lebl    Library of Libraries: Approach to Synthetic Combinatorial Library    Design and Screening of “Pharmacophore” Motifs PNAS 92: 5426-5430-   Bittle, J. L., Houghten, R. A., Alexander, H., Shinnick, T. M.,    Sutcliffe, J. G., Lerner, R. A., Rowlands, D. J. and Brown, F. 1982.    Protection against foot-and-mouth disease by immunization with a    chemically synthesized peptide predicted from the viral nucleotide    sequence. Nature 298:30-33.-   Harlow, E. & Lane, D. 1988. Antibodies: A laboratory manual, Cold    Spring Harbour Laboratory Press, Cold Spring Harbour, New York.-   Holder, A. A. and Freeman, R. R. 1981. Immunization against    bloodstage-rodent malaria using purified parasite antigens, Nature,    294:361-364.-   Hollingdale, M. R., Nardin, E. H., Tharavanij, S., Schwartz, A. L.    and Nussenzweig, R. S. 1984. Inhibition of entry of sporozoites of    Plasmodium falciparum and Plasmodium vivax sporozoites into cultured    cells; an in vitro assay of protective antibodies. J. Immunol.    132:909-913.-   Kaslow, D. C., Quakyi, I. A., Syin, C., Raum, M. G., Keister, D. B.,    Coligan, J. E., McCutchan, T. F. and Miller, L. H. 1988. A vaccine    candidate from the sexual stage of human malaria that contains    EGF-like domains. Nature 333:74-76.

The therapeutic agents may be delivered orally, in liposomes, by directinjection, or by controlled release from hydrophobic copolymers such asethylene-vinyl acetate, cyclodextrins, polysialic acid or surfactantsystems.

Suitable means are described in the following: (Ron, E. et al, 1993;);PNAS 90:4176-4180.

Alternatively, the receptor for Pid may be identified by protein-proteininteraction trap assays, such as the yeast two-hybrid system, describedin: (Chien, C-H. et al, 1991;); PNAS 88;9578-9582.

Inhibitors to this interaction may then be identified by furthercombinatorial chemistry, and duely optimised for inhibition bymutagenesis, using conventional methods. Suitable methods may be foundin the following references: Kirk McMillan, Marc Adler, Douglas S. Auld,John J. Baldwin, Eric Blasko, Leslie J. Browne, Daniel Chelsky, DavidDavey, Ronald E. Dolle, Keith A. Eagen, Shawn Erickson, Richard I.Feldman, Charles B. Glaser, Cornell Mallari, Michael M. Morrissey,Michael H. J. Ohlmeyer, Gonghua Pan, John F. Parkinson, Gary B.Phillips, Mark A. Polokoff, Nolan H. Sigal, Ronald Vergona, MarcWhitlow, Tish A. Young, and James J. Devlin Allosteric inhibitors ofinducible nitric oxide synthase dimerization discovered viacombinatorial chemistry PNAS 97: 1506-1511

-   Dustin J. Maly, Ingrid C. Choong, and Jonathan A. Ellman    Combinatorial target-guided ligand assembly: identification of    potent subtype-selective c-Src inhibitors PNAS 97: 2419-2424.-   R A P Lutzke, N A Eppens, P A Weber, R A Houghten, and R H A    Plasterk Identification of a Hexapeptide Inhibitor of the Human    Immunodeficiency Virus Integrase Protein by Using a Combinatorial    Chemical Library PNAS 92: 11456-11460.-   J J Burbaum, M H J Ohlmeyer, J C Reader, I Henderson, L W Dillard, G    Li, T L Randle, N H Sigal, D Chelsky, and J J Baldwin A Paradigm for    Drug Discovery Employing Encoded Combinatorial Libraries PNAS 92:    6027-6031-   D. T. O'Hagan et al. Microparticles in MF59, a potent adjuvant    combination for a recombinant protein vaccine against HIV-1 Vaccine    18(17):1793-1801 (2000)-   Jing Huang and Stuart L. Schreiber A yeast genetic system for    selecting small molecule inhibitors of protein-protein interactions    in nanodroplets PNAS 94: 13396-13401.-   Jay Parrish, Helen Metters, Lin Chen, and Ding Xue Demonstration of    the in vivo interaction of key cell death regulators by    structure-based design of second-site suppressors PNAS 97:    11916-11921.

The inhibitors may then be incorporated into therapeutic agents forreceptor occlusion, and tested for therapeutic efficacy using standardmethods.

-   Bittle, J. L., Houghten, R. A., Alexander, H., Shinnick, T. M.,    Sutcliffe, J. G., Lerner, R. A., Rowlands, D. J. and Brown, F. 1982.    Protection against foot-and-mouth disease by immunization with a    chemically synthesized peptide predicted from the viral nucleotide    sequence. Nature 298:30-33.-   Holder, A. A. and Freeman, R. R. 1981. Immunization against    bloodstage-rodent malaria using purified parasite antigens, Nature,    294:361-364.-   Kaslow, D. C., Quakyi, I. A., Syin, C., Raum, M. G., Keister, D. B.,    Coligan, J. E., McCutchan, T. F. and Miller, L. H. 1988. A vaccine    candidate from the sexual stage of human malaria that contains    EGF-like domains. Nature 333:74-76.    Diagnostic agent production and method of diagnosis Diagnostic agent    comprising antibody

Following expression of the Pid protein or a fragment thereof, methodsfor raising antibodies to the protein are well known in the art.Suitable methods may be found in the following references: Bittle, J.L., Houghten, R. A., Alexander, H., Shinnick, T. M., Sutcliffe, J. G.,Lerner, R. A., Rowlands, D. J. and Brown, F. 1982. Protection againstfoot-and-mouth disease by immunization with a chemically synthesizedpeptide predicted from the viral nucleotide sequence. Nature 298:30-33.

-   Harlow, E. & Lane, D. 1988. Antibodies: A laboratory manual, Cold    Spring Harbour Laboratory Press, Cold Spring Harbour, New York.-   Holder, A. A. and Freeman, R. R. 1981. Immunization against    bloodstage-rodent malaria using purified parasite antigens, Nature,    294:361-364.-   Hollingdale, M. R., Nardin, E. H., Tharavanij, S., Schwartz, A. L.    and Nussenzweig, R. S. 1984. Inhibition of entry of sporozoites of    Plasmodium falciparum and Plasmodium vivax sporozoites into cultured    cells; an in vitro assay of protective antibodies. J. Immunol.    132:909-913.-   Kaslow, D. C., Quakyi, I. A., Syin, C., Raum, M. G., Keister, D. B.,    Coligan, J. E., McCutchan, T. F. and Miller, L. H. 1988. A vaccine    candidate from the sexual stage of human malaria that contains    EGF-like domains. Nature 333:74-76.

These antibodies may be incorporated into a diagnostic agent and used indiagnosis according to conventional methods. The reader is directedtowards the following publications: Shuli Li, Gina Galvan, Fausto G.Araujo, Yasuhiro Suzuki, Jack S. Remington, and Stephen Parmley.Serodiagnosis of Recently Acquired Toxoplasma gondii Infection Using anEnzyme-Linked Immunosorbent Assay with a Combination of RecombinantAntigens Clin. Diagn. Lab. Immunol. 2000 7: 781-787.

-   Kerstin Hubert, Abel Andriantsimahavandy, Alain Michault, Matthias    Frosch, and Fritz A. Müühlschlegel Serological Diagnosis of Human    Cysticercosis by Use of Recombinant Antigens from Taenia solium    Cysticerci Clin. Diagn. Lab. Immunol. 1999 6: 479-482.-   Byoung-Kuk Na and Chul-Yong Song Use of Monoclonal Antibody in    Diagnosis of Candidiasis Caused by Candida albicans: Detection of    Circulating Aspartyl Proteinase Antigen Clin. Diagn. Lab. Immunol.    1999 6: 924-929.-   Felix Grimm, Friedrich E. Maly, Jian Lüü, and Roberto Llano Analysis    of Specific Immunoglobulin G Subclass Antibodies for Serological    Diagnosis of Echinococcosis by a Standard Enzyme-Linked    Immunosorbent Assay Clin. Diagn. Lab. Immunol. 1998 5: 613-616.-   Armando Reyna-Bello, Axel Cloeckaert, Nieves Vizcaííno, Mary I.    Gonzatti, Pedro M. Aso, Géérard Dubray, and Michel S. Zygmunt    Evaluation of an Enzyme-Linked Immunosorbent Assay Using Recombinant    Major Surface Protein 5 for Serological Diagnosis of Bovine    Anaplasmosis in Venezuela Clan. Diagn. Lab. Immunol. 1998 5:    259-262.-   Franççois Simondon, Isabelle Iteman, Marie Pierre Preziosi,    Abdoulaye Yam, and Nicole Guiso Evaluation of an Immunoglobulin G    Enzyme-Linked Immunosorbent Assay for Pertussis Toxin and    Filamentous Hemagglutinin in Diagnosis of Pertussis in Senegal Clin.    Diagn. Lab. Immunol. 1998 5: 130-134.-   I Bjerkas, M C Jenkins, and J P Dubey Identification and    characterization of Neospora caninum tachyzoite antigens useful for    diagnosis of neosporosis Clin. Diagn. Lab. Immunol. 1994 1: 214-221.-   M Theisen, J Vuust, A Gottschau, S Jepsen, and B Hogh Antigenicity    and immunogenicity of recombinant glutamate-rich protein of    Plasmodium falciparum expressed in Escherichia coli Clin. Diagn.    Lab. Immunol. 1995 2: 30-34.-   Laurens A. H. van Pinxteren, Pernille Ravn, Else Marie Agger, John    Pollock, and Peter Andersen Diagnosis of Tuberculosis Based on the    Two Specific Antigens ESAT-6 and CFP10 Clin. Diagn. Lab. Immunol.    2000 7: 155-160.

E. E. Zijlstra, N. S. Daifalla, P. A. Kager, E. A. G. Khalil, A. M.El-Hassan, S. G. Reed, and H. W. Ghalib rK39 Enzyme-Linked ImmunosorbentAssay for Diagnosis of Leishmania donovani Infection Clin. Diagn. Lab.Immunol. 1998 5: 717-720.

Diagnostic Agent Comprising Antigen

An effective antigenic component may be obtained as described above. Theantigenic component may then be incorporated in a diagnostic agent andused in diagnosis according to standard methods.

Suitable methods are described in the following documents: (Mills, C. D.et al, 1999;); Bull WHO 77:553-559; Felix Grimm, Friedrich E. Maly, JianLiu, and Roberto Llano Analysis of Specific Immunoglobulin G SubclassAntibodies for Serological Diagnosis of Echinococcosis by a StandardEnzyme-Linked Immunosorbent Assay Clin. Diagn. Lab. Immunol. 1998 5:613-616.

Armando Reyna-Bello, Axel Cloeckaert, Nieves Vizcaííno, Mary I.Gonzatti, Pedro M. Aso, Géérard Dubray, and Michel S. Zygmunt Evaluationof an Enzyme-Linked Immunosorbent Assay Using Recombinant Major SurfaceProtein 0.5 for Serological Diagnosis of Bovine Anaplasmosis inVenezuela Clin. Diagn. Lab. Immunol. 1998 5: 259-262.

Franççois Simondon, Isabelle Iteman, Marie Pierre Preziosi, AbdoulayeYam, and Nicole Guiso Evaluation of an Immunoglobulin G Enzyme-LinkedImmunosorbent Assay for Pertussis Toxin and Filamentous Hemagglutinin inDiagnosis of Pertussis in Senegal Clin. Diagn. Lab. Immunol. 1998.5:130-134.

-   Bjerkas, MC Jenkins, and JP Dubey Identification and    characterization of Neospora caninum tachyzoite antigens useful for    diagnosis of neosporosis lin. Diagn. Lab. Immunol. 1994 1: 214-221.-   M Theisen, J Vuust, A Gottschau, S Jepsen, and B Hogh Antigenicity    and immunogenicity of recombinant glutamate-rich protein of    Plasmodium falciparum expressed in Escherichia coli Clin. Diagn.    Lab. Immunol. 1995 2: 30-34. Laurens A. H. van Pinxteren, Pernille    Ravn, Else Marie Agger, John Pollock, and Peter Andersen Diagnosis    of Tuberculosis Based on the Two Specific Antigens ESAT-6 and CFP10    Clin. Diagn. Lab. Immunol. 2000 7: 155-160.-   E. E. Zijlstra, N. S. Daifalla, P. A. Kager, E. A. G. Khalil, A. M.    El-Hassan, S. G. Reed, and H. W. Ghalib rK39 Enzyme-Linked    Immunosorbent Assay for Diagnosis of Leishmania donovani. Infection    Clin. Diagn. Lab. Immunol. 1998 5: 717-720.    Materials and Methods

1. Construction of Cosmid Library of P. yoelii Genomic DNA.

High molecular weight genomic DNA was isolated from an asynchronousculture of P. yoelii and partially cleaved with Sau3A to yield fragmentsof 30-50 kb. These were dephosphorylated with calf intestinal alkalinephosphatase and ligated into the cosmid vector SuperCos1 (Stratagene)previously digested with BamH1. An aliquot of the ligation mixture waspackaged with Gigapack III XL packaging extract (Stratagene) andtransduced into XL1-Blue MR (Stratagene) Recombinant cosmids wereselected on ampicillin, pooled and amplified once.

2. Primary Screening of Cosmid Clones for Invasion of Cultured COS-7Cells.

For invasion assays, a logarithmic phase culture of E. coli harbouringthe cosmids were seeded onto COS-7 cells at a multiplicity of infectionof 10, and invasion assays performed essentially as described. After 5 hof incubation the cells were washed extensively and fresh mediumcontaining 250 μg/ml gentamicin was added to kill non-invading bacteria.After an overnight incubation, the cells were washed 10× with PBS.Invading bacteria were released by gentle lysis of the COS-7 cells withPBS/0.05% saponin, and scored by ampicillin selection on LB-plates.

3. Immunofluorescence and Electron Microscopy Studies.

Invasion assays were performed as above. The cells were then washedextensively, and fixed with 3.7% paraformaldehyde. The cells werepermeabilised at room temperature for 30 min with PBS/0.05% saponin,incubated with 5% non-fat dry milk in PBS. After 1 h at roomtemperature, the cells were washed 3× witch PBS and incubated with arabbit polyclonal antibody to the E. coli K-12 strain C600 (Dako Ltd).This antibody cross-reacts with other K-12 strains. Following incubationfor 2 h, the cells were washed with PBS and incubated with rabbitFITC-labelled IgG (Sigma) for 1 h, washed extensively with PBS andmounted for fluorescence microscopy on a Zeiss microscope. The resultsare illustrated in FIG. 2A (negative control) and FIG. 2B infectionswith E. coli containing invasion cosmid clone.

Cells may also be counter stained with TRITC-phalloidin to determinebacteria and actin polymer colocalisation.

In order to obtain electron micrographs of COS-7 cells invaded bytransformed E. coli, invasion assays were carried out as above, and theCOS-7 cells then washed extensively with PBS before fixing with 4%glutaraldehyde overnight at 4° C. The fixed cells were sectioned,stained with osmium tetroxide and viewed with a transmission electronmicroscope. The results are shown in FIG. 2C.

4. Transposon Mutagenesis of Identified Invasive Clones, and SequenceAnalysis.

Transposon mutagenesis was performed using the Tn1000γδ, essentially asdescribed, with minor modifications as follows.

Two invasive clones Invcos11 and Invcos18 were transformed intocompetent MH1638 donor cells. Single colonies were grown to log phase inLB/100 μg per ml ampicillin/50 μg per ml methicillin. Recipient cells,HB101 were grown in LB. 2 ml of donor culture were spun and resuspendedwith 15 ml of LB in Falcon tubes. After centrifuging for 10 min at 4500rpm, the cell pellet was resuspended with 1 ml of recipient HB101culture. This mating mixture was spread on LB agar plates (withoutantibiotics) and incubated for 2 h at 37° C.

The cells were washed with 15 ml LB broth and centrifuged as above.After a second spin, the pellet was resuspended in 1 ml LB broth. 100 μlof this was plated on LB plates supplemented with 100 μg/ml ampicillin,501 g/ml methicillin and 1001 g/ml streptomycin. After an overnightincubation at 37° C., 50 isolated colonies were picked, grown inLB/ampicillin/methicillin/streptomycin (LB/amp/meth/strep), and used ininvasion assays as described above.

Invading colonies were scored by plating saponin-lysed COS-7 cells onLB/amp/meth/strep. FIG. 3 a shows plates obtained in plate scoring afterinvasion assays using a wild type and a Tn1000γδ-inserted clone.

FIG. 3 b provides a bar chart displaying the results of plate scoringfollowing invasion assays using Tn1000γδ-inserted clones TM1 and TM5.Wild type Invcos11 and Invcos18 were used as positive controls forinvasion, while HB101 or XL1 Blue transformed with pMAL-p2 (New EnglandBiolabs) grown on LB agar or LB/amp agar acted as negative controls.

Non-complementing clones identified from this assay were purified andsequenced using the following primers: γ CCTGAAAAGGGACCTTTGTATACTG (SEQID No 13) δ AGGGGAACTGAGAGCTCTA (SEQ ID No 14)The sequence of the inserted region, shown in FIG. 4A, was obtained bycontig assembly of the sequences from the transposon mutants.Antigenic Responses to PidMethods

1. Sub-Cloning of pid and expression of Pid-Myc fusion protein. pid wasprepared as a PCR amplimer from invcos18 with HindIII and Bg1restriction sites at the 5′ and 3′ ends respectively. The amplimer wassub-cloned into the expression vector pROlar A122(Clontech) whichincludes a Myc epitope tag. Colonies were maintained on LB/kanamycinplates at 37° C. A 50 μl aliquot of an over night broth culture was usedto inoculate a 5 ml broth and this was incubated to 0.4-0.6 OD₆₀₀, atthis point the culture was induced with 5 μl 100 mM IPTG and 53 μl 15%arabinose. The culture was incubated for 3 hours at 37° C.

2. Pid preparation. Concentration of Pid was enhanced using c-Mycmonoclonal antibody-agarose beads (Clontech). Following induction cellswere washed in ice-cold PBS, the pellet was then re-suspended in lysisbuffer and frozen at −70° C. The sample was then thawed on ice and 2041c-Myc Mab was added, vortexed briefly and mixed on a rotating platformfor 40 min at 4° C. The preparation was then washed 3 times in ice coldPBS, with microcentrifugation at 1200 g for 1 min.

3. Patient sera. Serum was collected from 8 patients admitted to theRoyal Free Hospital with malaria. Plasmodium falciparum infection anddegree of parasitaemia confirmed. Country of origin was noted.

4. Western blot analysis. Multiple lanes of Pid preparation wereseparated by 12% SDS-PAGE prior to electrophoretic transfer to Hybond-C(AmershamPharmacia), transfer was for 2 h at 10V (Novablot,AmershamPharmacia). The resultant blots were probed with each patientserum at a dilution of 1/100 aid visualised using the ECL WesternBlotting reagents (AmershamPharmacia)

Results

Five out of eight patient sera tested recognised a protein band thatco-migrated with the Pid-Myc fusion product. These sera did notrecognise a band of this molecular weight when cells transformed withpROlar containing an unrelated non-Plasmodium sequence were tested. Thecountry of origin of each patient and levels of parasiteamia are shownin Table 1.

CONCLUSION

The preparation of E. coli cells with induced expression of Pid did showthe presence of an antigenic moiety of the predicted molecular weight in5/8 sera tested. The patients all had marked parasitaemia and were fromsub-saharan Africa. This data indicates that patients exposed toPlasmodium falciparum do raise an immune response to Pid and thereforethis may form the basis of a serological assay for the detection of thisinfection. TABLE 1 Parasitaemia Country Positive on Patient % of originWestern blot 1 3.8 Ghana + 2 <1.0 Ghana ++ 3 <1.0 Ghana − 4 1.2 Ghana−/+ 5 4.5 Tanzania ++ 6 <1.0 Nigeria − 7 <1.0 Ghana + 8 <1.0 Nigeria ++PCR Amplification of Pid from Patient Sera.Method

1. PCR amplification of Did: Using genomic DNA from falciparum T9-96 anested PCR protocol was using the following primers; first round 5′ ATGCTG ATG TTG CTA CGG 3′, pfpid2, 5′ATC TTC CTG CAT TGC TCA CGC 3′ andSecond round primers, pfpid3 5′ CTT GGA ATG AGG TTG TTT G 3′ and pfpid45′ AAT CCT CGA CGC CTA ACG 3′ (FIG. 1). The optimised reaction mixturefor the first round PCR was, 1×NH₄Cl₂ stock buffer (Biolline, UK), 5 mMMgCl₂, 1 μM pfpid1, 1 μM pfpid2, 100 μM dNT2, 2.5 IU Taq polymerase(Bioline, UK). Cycling conditions were 95° C. for 3 min, 45 cycles of92° C. 30 sec, 50° C. 30 sec, 72° C. 30 sec followed by a cycle of 72°C. 5 min. For the nested PCR 2 μl of the amplimer preparation from thefirst round PCR was added to the reaction mix; 1× NH₄Cl₂ stock buffer(Bioline, UK), 5 mM MgCl₂, 1 μM pfpid3, 1 μM pfpid4, 200 μM dNTP, 2.5 IUTaq polymerase (Bioline, UK). Cycling conditions were 95° C. for 3 min,45 cycles of 92° C. 30 sec, 55° C. 30 sec. 72° C. 30 sec followed by acycle of 72° C. 5 min.

2. Patient samples. Whole blood was collected from 8 patients admittedto the Royal Free Hospital with malaria. Fresh whole blood wascentrifuged at 3,000 g for 5 mins. The serum was removed and the redcells stored at −20 CC. Plasmodium falciparum infection and degree ofparasitaemia confirmed. Country of origin was noted.

3. PCR amplification of pid from patient samples: DNA was extracted fromthe red cell pellet, according to the manufacturers instructions usingthe Wizard® Genomic DNA Purification Kit (Promega, UK). 5 μl of theresultant genomic DNA was then amplified according to the optimisedprotocol in section 1. PCR amplimers were visualised on a 2% agarose gelcontaining ethidium bromide.

4. Sequence analysis of PCR amplimers from patient samples: PCRamplimers of the predicted molecular weight were purified using theWizard® PCR Purification Kit (Promega, UK). Amplimers were thensubmitted for sequencing to Cambridge Biosciences (now Cytomyx, UK).

Results

FIG. 6 shows the results of PCR amplification of pid from patientsamples; 17/20 samples gave positive results, this represents positiveresults from all patients tested, 3 patients had one negative result.Patient details and PCR results are summarised in Table 2.

Sequencing was performed using amplimers from the PCR reactions givingthe strongest results, samples 4, 5 and 10. Analysis of the similarityof these sequences to pid was performed using PILEUP (GCG, MRC-HGMP UK),samples 4 and 5 came from the same patient, all 3 samples show a highdegree of similarity to pid (FIG. 7).

CONCLUSION

This data provides evidence that pid can be amplified from patients withconfirmed Plasmodium falciparum infection and thus may be a suitabletarget for a diagnostic test. The PCR amplimers investigated forsimilarity to pid showed a high degree of homology, confirming theiridentity as pid. TABLE 2 Sample Parasitaemia Patient Number Country oforigin % pid PCR A 1 Ghana 3.5 + 2 3.8 + 3 <1.0 − B 4 Ghana <1.0 + 5 0 +14 0 − C 6 Ghana <1.0 + 7 <1.0 + 8 0 + 9 0 + D 10 Tanzania 1.2 + 114.5 + 12 <1.0 + 13 0 − E 15 Nigeria <1.0 + 16 <1.0 + F 17 ? <1.0 + 18<1.0 + G 19 ? <1.0 + 20 1.5 +pid mediated invasion of human red blood cellsMethods

1. Bacterial cell lines: E. coli K12 strain XL1-Blue MR transformed witheither invcos18, pROLAR-pid or native pROLAR were used. These constructsare described above.

2. Human red blood cells (rbc): 10 ml freshly drawn blood wastransferred to a tube containing lithium heparin. The blood donor had nohistory of travel to an area endemic for malaria in any form and had noknown exposure to malaria. The rbc were separated by centrifugation at3,000 g for 5 min and washed 3 times in DMEM/10% foetal bovine serum(FBS) by microcentrifugation at 5000 g.

3. Invasion assay: Transformed E. coli were incubated with red bloodcells at a ratio of 10:1 (85×10⁵ bacteria: 8.5×10⁵ rbcs) for 3 h at 37°C. in 5% CO₂. The cells were then washed 3 times in DMEM/10% FBS bymicrocentrifugation at 5000 g. The washed cells were resuspended inDMEM/FBS, 200 μg/ml gentamicin was added and the preparation wasincubated overnight at 37° C. in 5% CO₂. Following incubation the cellswere washed 3 times in DMEM/10% FBS by microcentrifugation at 5000 g andthe rbc were lysed by resuspension in sterile distilled water. 50 μl ofthe lysates was spread on LB agar plates containing kanamycin, lysateswere plated in duplicate.

Plates were read after 24 h and the number of colonies counted by 2independent observers.

Results

Colony counts are shown in Table 3.

CONCLUSION

The data presented in Table 3 indicate that invasion of human red bloodcells by E. coli can be mediated by the presence of pid alone, however,invasion is enhanced by the presence of the complete cosmid, suggestingthe presence of further genes that contribute to the invasive process.TABLE 1 pid mediated invasion of fresh human red blood cells Duplicatecolony counts Construct Observer 1 Observer 2 pROLAR 46/48 51/47pROLAR-pid 88/90 97/99 Invcos18 780/881 847/743

INTERACTION BETWEEN PID AND THE RHO GTPASES

cDNAs encoding human Cdc42, Rac1 and RhoA were amplified by PCR with thefollowing set of primers:

Cdc42: sense (cag gaa ttc cag aca att aag tgt gtt g); antisense (cag gtcgac tta gaa tat aca gca ctt cc). Rac1: sense (cag gaa ttc cag gcc atcaag tgt gtg); antisense (cag gtc gac cta caa cag gca ttt tct c). RhoA:sense (cag gaa ttc gct gcc atc cgg aag aaa ctg); antisense (cag cgt cgactc aca aga caa ggc aac c). All three GTPases were digested with EcoRIand SalI and ligated into the same sites in the activation domain vectorpAD-GAL4 (Stratagene). Pid was also amplified (sense: cag gga att catgct gat gtt gct ac); antisense (cag cgt cga cct aga tct tcc tgc),digested with the above enzymes and ligated into the binding-domainvector pBD-GAL4 (Stratagene). All 4 legations were used to transformcompetent DH5α cells and selected on LB agar/ampicillin plates.Transformants were screened for inserts, and these inserts wererestriction-mapped to determine whether they were n the rightorientation, with respect to the GAL4 fusion domains. The new plasmidswere designated AD-Cdc42, AD-Rac1, AD-RhoA and BD-Pid for the respectiveproteins.

To determine interaction, the AD-GTPase constructs were eachcotransformed with BD-Pid into competent yeast cells YRG-2 (Stratagene).These were then plated on synthetic dextrose agar plates(SD/-His-Leu-Trp). This medium lacks the amino acids leucine, (whichselects for activation domain-GTPase constructs), tryptophan (whichselects for binding domain-Pid) and histidine, which selects for the HISreporter gene. After 3-7 days incubation at 30° C., colonies wereisolated and grown in SD liquid medium for 3 days at 30° C. in a shakingincubator.

Observation: Interaction between Pid and the RhoA GTPases was scoredbased on both amino acid prototrophy and the expression of the HISreporter gene. However, only Cdc42 appeared to be definitely interactingwith Pi-d.

Subcellular Localization of PID

Using a reporter protein, the green or red fluorescent protein, it isoften possible to track the location of a protein using cells underculture conditions. To do this, Pid was PCR-amplified With the primers:cag gga att cat gct gat gtt gct ac (sense) and antisense (cat gct cgagat ctt cct gca ttg ctc ac) and digested with EcoRI and XhoI. It wasthen ligated into the EcoRI/SalI sites of pDsRed-N1 (Clontech), at theN-terminus and in-frame with the red fluorescent protein. Transformantswere derived from DH5a cells, which were selected on kanamycin LB agarplates. Plasmid DNA was purified and the presence and orientation of Pidinsert were determined by restriction mapping. The plasmid wasdesignated pPid-DsRed.

HeLa cells (˜10⁴ cells) were seeded on Permanox chamber slides and grownin Dulbeccos Modified Eagles Medium (DMEM) supplemented with 10% foetalbovine serum, antimycotics and antibiotics, at 37° C. under 5% CO₂. Atabout 80% confluence the cells were rinsed and then incubated with freshmedium 1-2 hr before transfection. pPid-DsRed (5 μg) was diluted inOptiMEM medium to a final volume of 100 μl. 10 μl liposomes (DOSPER,Roche) was also diluted to the same volume. Both plasmid DNA andliposome dilutions were mixed and incubated at room temperature for 30min to allow complex formation. The mixture was then transferred with apipette onto the HeLa cells and incubation continued for 6-12 hrs. FreshDMEM was then added and the cells incubated for another 30 hrs. Afterthis time, the cells were washed 2× with PBS, and fixed with 3.7%paraformaldehyde for 30 mins at room temperature. They were then mountedin Vectashield Mounting Medium with DAPI for nuclear counter-staining(Vector Laboratories) and viewed under a Nikon Eclipse E800 fluorescentmicroscope. Images were acquired from 2 micron sections with the Bio-RadRadiance 2100 confocal microscope.

Observation: In HeLa cells transfected with pDsRed-N1, fluorescence wasobserved throughout the entire cell, both in the cytosol and nuclearlumen. In contrast, in cells transfected with pPid-DsRed, fluorescenceappeared to be confined within granules (possibly secretory) orperipheral vesicles that were in juxtaposition to the plasma membrane.

ACTIN DEPOLYMERIZATION IMPEDES CELL INVASION BY PID-TRANSFORMED BACTERIA

In earlier experiments it was observed by double staining withFITC-labelled antibodies and TRITC-phalloidin, that bacterial cellstransformed with the invasion cosmid induced actinnucleation/polymerisation at foci of cell entry. This suggested therecruitment of cytoskeletal components in the invasion process. Takingthis a step further, it was considered whether actin depolymerisationhad any effect on the invasion process. To address this, 10⁴ HeLa cellswere seeded onto Thermanox cover slips and cultured in DMEM as above.Before invasion assays, the HeLa cells were washed 2× with PBS and thenincubated with fresh medium lacking antibiotics. For inhibition,cytochalasin B was added to the cells at a concentration of 0.5-1 μg/ml,2-5 mins before invasion assays were initiated. Bacterial cellscontaining pA15-Pid (Pid subcloned into pROLar.A122), pGEX-Pid (Pidsubcloned into pGEX-5×1), Incos18 (wild-type invasion cosmid), In18-M1(mutant cosmid) or untransformed HB101 were then added to the HeLa cellculture at an m.o.i of 10. To control for invasion efficiency,cytochalasin B was omitted in parallel assays with the transformedbacteria. After 3-5 hr, the cells were washed 5× with PBS and thenincubated with DMEM supplemented with −200 μg/ml gentamicin and leftovernight in the incubator. The cells were then washed with PBS (5×),fixed with 3.7% paraformaldehyde and permeabilized with PBS/0.05%saponin for 30 min at room temperature. They were incubated for 1 hr atroom temperature with. PBS/5% non-fat dry milk and then with anti-E.coli antibody diluted in PBS/5% non-fat dry milk. They were rinsed 2×with PBS and incubated again for 1 hr with anti-rabbit IgG-FITCconjugate alone or with TRITC-phalloidin. The cells were Lhen washedextensively with PBS, and mounted for fluorescence microscopy.

Observation: HeLa cells treated with cytochalasin B were refractory toinvasion by pA15-Pid or pGEX-Pid-transformed bacteria.Incos18-transformed cells showed a localization of the bacterial cellsat the periphery of the cytochalasin B-treated HeLa cells if they werepresent. This showed the uncoupling of bacterial cell attachment to theHeLa cells from the invasion step. HeLa cells untreated withcytochalasin B were efficiently invaded by Incos18, pGEX-Pid andpA15-Pid but not by In18TM1 or HB101.

Corresponding Pid Sequence in Cryptosporidium

Cryptosporidium parvum oocysts were obtained from Moredun ScientificLtd, Scotland. Approximately 100,000 oocysts were centrifuged andrespuspended in 200 μl of 50 mM Tris-HCl pH 8.0, 5 mM EDTA, 50 mM NaCl,0.5% Sarkosyl, 100 ug/ml proteinase K. The cells were incubated for 2hrs at 60° C. in a PCR machine. The lysate was then extracted 2× withphenol-chloroform-isoamyl alcohol. Genomic DNA was precipitated with 0.1volume of 3M sodium acetate pH 5.2 and 2.5 volumes of ice-cold 95%ethanol. After 30 mins at −20° C., the DNA was pelleted bycentrifugation at 13,000 rpm for 30 mins, washed with 70% ethanol andair-dried. The DNA pellet was resuspended in 100 μl 10 mM Tris/1 mM EDTApH 8.0. For FCR, the following primers were used: sense, CGA GAA TTC ATGCTA ATG TTG CTA CGG; antisense, CGA AGC TTC TAG ATC TTC CTG CAT TGC. Thecycling parameters were as follows: Initial denaturation at 94° C. for 3mins, then 30 cycles at: 94° C. for 30 secs, 55° C. for 30 secs and 1min at 68° C. A final extension at 68° C. was done for 10 mins. The PCRproduct was ligated into the T-vector pCR2.1 (Invitrogen), plasmid DNAwas purified from recombinants and sequenced.

Observation: Sequencing revealed sequence ID NO:28, which is virtuallyidentical to the Pid nucleotide sequence from plasmodium yoeli.

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FIG. 4 legends

A. Nucleic acid and translated amino acid sequence of pid and adjacentsequences. Upstream is ORF3, part of the osa operon in pSa; downstreamof pid is the 5′ UTR of the antirestriction gene ArdC. Transposoninsertion sites are indicated by arrow heads (□). Termination signalsare indicated with asterisks. Sequences upstream of transposon insertionsites were obtained with the γ primer while downstream sequences wereobtained with the δ primer of the Tn1000 transposon. Interveningsequences were obtained by primer-walking across the transposoninsertion sites, and verified by sequencing several other clonesincluding the wild-type cosmid construct Invcos18.

B. Schematic representation of pid as well as upstream and downstreamsequences. The direction of transcription is indicated by an arrow. Theputative Cdc42/Rho GTPase-interacting sequence (the CRIB domain) SEQ IDNO:6 is in bold print and is compared with that of Ste20/S cerevisiae(Acc # Lb4655); human WASP (Acc # NM000377); Cdc42 effector proteinMSE55 (Acc # XM_(—)001058.1); p65^(PAK)-α/rat (Acc # NM017198);C09B8.7/C.elegans (Acc # U29612) and SHK1/S. pombe (Acc # AL034433).

1. An antigenic component, for use in a vaccine capable of promotingproduction in a subject of an antibody specific to the antigeniccomponent, which antibody is capable of specifically binding to the Pidprotein having the amino acid sequence in Seq ID No.1.
 2. An antigeniccomponent, for use in a vaccine capable of promoting production in asubject of an antibody specific to the antigenic component, whichantibody is capable of specifically binding to the Pid protein havingthe amino acid sequence in Seq ID No.1, wherein the antigenic componentcomprises a component selected from the Pid protein having the aminoacid sequence in Seq ID No 1 a peptide fragment of the Pid proteinhaving the amino acid sequence in Seq ID No 1, and a variant of the Pidprotein or peptide fragment thereof which does not substantially affectits antigenicity.
 3. An antigenic component according to claim 2 whichis preparable from an apicomplexan parasite.
 4. An antigenic componentaccording to claim 3 wherein the apicomplexan parasite is of a genusselected from the following :Eimeria; Isospora; Toxoplasma; Hammondia;Cystoisospora; Sarcocystis; Besnoitia; Frenkelia; Cryptosporidium;Plasmodium; Babesia; and Theileria.
 5. An antigenic component accordingto claim 4 wherein the apicomplexan parasite is of the genus Plasmodium.6. An immunogen comprising an antigenic component according to claim 1coupled to an immunogenic component.
 7. A vaccine comprising animmunogen according to claim 7 and an adjuvant.
 8. A vaccine comprisinga polynucleic acid, which encodes an antigenic component according toclaim
 1. 9. A vaccine according to claim 8 wherein the polynucleic acidfurther comprises a eukaryotic promoter for controlling expression ofthe sequence encoding the antigenic component.
 10. A vaccine accordingto claim 8 which is suitable for use in a human subject.
 11. A vaccineaccording to claim 10 which is suitable for use against human malariacaused by a parasite selected from: P. falciparum; P. ovale; P. vivax;and P. malariae.
 12. A therapeutic agent comprising a component whichcomponent is capable of competing with a protein having the amino acidsequence in Seq ID No 1 in a specific binding assay.
 13. A diagnosticagent comprising an antibody, which antibody is capable of specificallybinding to the Pid protein having the amino acid sequence in Seq IDNo
 1. 14. A diagnostic agent comprising an antigenic component accordingto claim
 1. 15. A pharmaceutical composition which comprises a proteincomprising the amino acid sequence in SeqID No 1, or a peptide fragmentthereof.
 16. A pharmaceutical composition which comprises a polynucleicacid encoding a Pid protein having the amino acid sequence in SeqID No1, or a fragment thereof.
 17. A pharmaceutical comprising an antibody,which is capable of specifically binding to the Pid protein having theamino acid sequence in Seq ID No
 1. 18. A method for prophylactic ortherapeutic treatment of a disease caused by an apicomplexan parasite,which comprises administering to a subject an effective amount of amedicament comprising an antigenic component capable of promotingproduction in a subject of an antibody specific to the antigeniccomponent, which antibody is capable of specifically binding to the Pidprotein having the amino acid sequence in Seq ID No.
 1. 19. A method forprophylactic or therapeutic treatment of a disease caused by anapicomplexan parasite, which comprises administering to a subject aneffective amount of a medicament comprising an antigenic componentcapable of promoting production in a subject of an antibody specific tothe antigenic component, which antibody is capable of specificallybinding to the Pid protein having the amino acid sequence in Seq ID No.1, wherein the antigenic component comprises a component selected fromthe Pid protein having the amino acid sequence in Seq ID No 1 a peptidefragment of the Pid protein having the amino acid sequence in Seq ID No1, and a variant of the Pid protein or peptide fragment thereof whichdoes not substantially affect its antigenicity.
 20. A method accordingto claim 19 wherein the disease is selected from: malaria; coccidiosis;theileriosis; cryptosporidiosis; isosporiasis; blastocystosis;babesiosis; anaplasmosis; sarcosporidiosis; toxoplasmosis; andsarcocystosis.
 21. A method according to claim 20 wherein theapicomplexan parasite is of the genus Plasmodium.
 22. A method accordingto claim 21 wherein the apicomplexan parasite is one selected from thefollowing: Plasmodium falciparum; Plasmodium vivax; Plasmodium ovale;and Plasmodium malariae.
 23. A method for prophylactic or therapeutictreatment of malaria, which comprises administering to a human subjectan effective amount of a vaccine comprising an antigenic componentselected from the Pid protein having the amino acid sequence in Seq IDNo 1 a peptide fragment of the Pid protein having the amino acidsequence in Seq ID No 1, and a variant of the Pid protein or peptidefragment thereof which does not substantially affect its antigenicity.24. A method for diagnosing apicomplexan infection in a subject, whichcomprises: (i) obtaining from the subject a sample of body fluid; and(ii) testing the sample by contacting therewith an antibody capable ofspecifically binding to the Pid protein having the amino acid sequencein SeqID No
 1. 25. A method for diagnosing apicomplexan infection in asubject, which comprises: (i) obtaining from the subject a sample ofbody fluid; and (ii) testing the sample by contacting therewith aprotein comprising the amino acid sequence in SeqID No 1, or a peptidefragment thereof.
 26. A method for diagnosing apicomplexan infection ina subject, which comprises: (i) obtaining from the subject a sample ofbody fluid; and (ii) testing the sample by contacting therewith apolynucleic acid encoding a Pid protein having the amino acid sequencein SeqID No 1, or a fragment thereof.
 27. A method for prophylactic ortherapeutic treatment of a disease caused by an apicomplexan parasite,which comprises administering to a subject an effective amount of amedicament comprising an inhibitor of Pid protein-Cdc42 interaction. 28.An in vitro method for diagnosing apicomplexan infection in a subject,which comprises: (i) obtaining from the subject a nucleic acidcontaining sample; and (ii) testing the sample for the presence ofnucleic acid sequence characteristic of Pid.
 29. A method according toclaim 28, wherein the apicomplexan is of the genus Plasmodium.
 30. Amethod according to claim 28, wherein sample comprises red blood cells.31. A method according to claim 28, wherein the nucleic acid sample isamplified prior to testing.